The invention relates to proteinase inhibitors and in particular novel proteinase inhibitors, methods for producing such inhibitors and products and processors including such inhibitors.
Proteinases are enzymes that break down proteins, their substrate specificity varies considerably and therefore does not form a basis for the purpose of classification. Rather, typically, these enzymes are classified according to the nature of the catalytic reaction that each undertakes. Thus proteinases are divided into four groups termed serine proteinases, cysteine proteinases, aspartic proteinases and metalloproteinases. Serine proteinases and cysteine proteinases are both widespread and diverse and are found in both prokaryotic and eukaryotic organisms, including plants and animals. In contrast, aspartic proteinases seem to be found only in eukaryotic organisms. Since these enzymes are used to break down protein the origin and/or the location of the enzymes determines whether they are beneficial or detrimental to a given organism. For example, where the enzymes are used by pathogens or parasites or pests they are typically used to break down host cell tissue and are therefore detrimental.
Pathogens, parasites or pests such as bacteria, fungi, plants, insects, nematode worms etc produce proteinases which break down host cell tissue to the detriment of the host.
For example, annual global crop losses caused by fungi exceed a thousand million pounds. The pathogen, Botrytis cinerea is of major economic importance because it causes disease in thirty crop plant species, with serious losses incurred in the glass house, in viticulture and as a result of post-harvest disease of fruit and vegetables. This major pathogen can be overcome with fungicides but unfortunately, there are disadvantages associated with the use of fungicides in order to control it. These include a financial burden associated with use of the fungicide, the potential environmental hazard arising from the use of toxic fungicides, with attendant consumer concern, and major problems of pathogen resistance to fungicides. In addition, many fungicides are effective against only a limited range of pathogenic fungi.
The above disadvantages are also common to the use of synthetic agents manufactured against other pathogens, parasites or pests such as insects, or specific insects, bacteria or specific bacteria and other eukaryotic organisms including, but not limited to: protozoa such as amoebas, intestinal flagellates and ciliates, haemoflagellates, such as leishmania or trypanosomes, sporozoa, such as those responsible for malaria, arthropod-borne organisms; helminths such as trematodes or flukes, cestoidea, acanthocephala, nematodes, trichuris, trichinella, hook worms, filariae, spiruroids; arthropods such as acarina or mites, ticks heteroptera, lice, flees, diptera such as disease-carrying flies including mosquitos, maggots and myiasis.
Inhibition of proteinases is known to occur naturally following pathogen infection. For example, it has been shown that following infection by Phytophthora infestans varieties of tomato able to resist the fungus show increased levels of proteinase inhibitors (1). This relationship between resistance and the capacity to produce proteinase inhibitors has been used to good effect in the control of pathogen, pests and parasitic diseases. For example, in the most relevant prior art known to the applicant, plant pests are controlled by recombinantly introducing a proteinase inhibitor, animal-derived egg white cystatin, into a selected monocotyledon such as a cereal, forage or turf grass, or a dicotyledon such as a vegetable, tube, or sugar crop, (EP 0 348 348). Similarly, plant nematode pests have been controlled using a proteinase inhibitor, plant-derived cowpea trypsin inhibitor, which has been recombinantly introduced into tobacco, tomato, cotton, oilseed rape, vegetable crop or ornamental plants (EP 0 502 730). In addition, it has been suggested that proteinase inhibitors can be used as anti-parasitic proteins which ideally can be administered to a host species either in a medicament or a food (UK Patent Application No. 94 03819.7).
It is therefore known to use proteinase inhibitors to neutralise the effects of proteinases and so combat the effects of pathogens, parasites or pests. In particular, it is known to transgenically produce plants which are provided with a specific proteinase inhibitor, such as a cysteine proteinase inhibitor.
However, it is the object of the present invention to provide a modified proteinase inhibitor which has greater efficacy than that of its unmodified counterpart or the natural proteinase inhibitor; or alternatively to synthetically manufacture and improve a proteinase inhibitor so as to provide, in one embodiment a hybrid proteinase inhibitor.
In one aspect of our invention we have focused on the group of proteinase inhibitors known as cystatins. The protein sequences of approximately 25 cystatins are known. It is possible to undertake alignment studies of these sequences in order to provide a basis for identifying structural similarities. It has been suggested that there are sufficient differences between plant and animal cystatins to justify separate classification of the two, indeed, a comparison of a plant cystatin, Oryzacystatin I Oc-I [DNA sequence structure shown in FIG. 3, SEQ ID NO:30], and an animal cystatin, egg white cystatin, reveals a significant number of differences showing that overall amino acid conservation is not high. Moreover, there are significant differences in the binding properties of animal and plant cystatins. Thus the dissociation constant Ki varies, for example, egg white cystatin has a Ki of 5xc3x9710xe2x88x9212M, whereas the plant cystatin, Oc-I, (derived from rice), has a Ki of 3xc3x9710xe2x88x928M.
Alignment data of a number of cystatins (SEQ ID NO:1-28) is shown in FIG. 1. The amino acids are numbered 1-181. It can be seen that there is a conserved inhibitory site at alignment amino acids 100-104, represented by the motif QVVAG (SEQ ID NO:56) or QLVAG (SEQ ID NO:57). In addition, it can be seen that there is a conserved PW motif at alignment amino acids 160-161.
This conservation occurs in approximately two thirds of the known sequence structures and is thought from structural studies to be involved in the functioning of the protein and thus for inhibition of proteinases. However, some cystatins with low Ki values do not possess this PW motif therefore its importance in cystatin function is unclear.
Other works have recombinantly manufactured novel cystatins. For example, the human cysteine proteinase inhibitor cystatin C, which participates in the intracellular catabolism of proteins and peptides, in the proteolytic conversion of prohormones, in the extracellular degregation of collagen and in the penetration of normal tissues with malignant cells, has been altered. Workers have modified cystatin C so that one or more amino acids at positions 5-17, 55-59 and/or 68 have been replaced by other amino acids thus retaining the total 120 amino acids in the sequence structure. Modifications were undertaken in order to provide an animal-derived cystatin C considered to have constant activity. (WO 88/09384).
We have found, surprisingly, that site-directed modification of a plant cystatin such as, for example, Oryzacystatin I (Oc-I) can improve its binding properties and thus improve the efficacy of the enzyme in inhibiting proteinases. The site-directed modification involves elimination of the amino acid aspartic acid at position 86 of the amino acid sequence structure of the plant cystatin, this elimination improves the Ki 13 fold, that is to 2.3xc3x9710xe2x88x929M. This modification is represented by elimination of aspartic acid (symbol D) at position 163 of the alignment amino acids shown in FIG. 1.
Clearly, this improvement in plant cystatin Ki does not exceed animal cystatin Ki, and particularly egg white cystatin. However, there is growing concern about the liberal approach to cross species transgenics. That is to say the introduction into one species of genes wholly from another species, such as for example, the introduction into plants of human genes encoding human proteins and visa versa. Until our understanding of the consequences of genetic manipulation is complete is would seem prudent to err on the side of caution and thus adopt a more rational approach to genetic manipulation. Thus, plant breeders throughout the world would prefer to combat plant diseases using plant derived proteins, or alternatively, proteins which are significantly similar to plant proteins such that their structural, biochemical and physiological functions are either the same as, or substantially similar to, or consistent with, that of plant proteins. This letter category includes, but is not limited to hybrid molecules.
Further, many animal cystatins with lower Ki""s have several disulphide bonds which are not found in plant cystatins so far characterised. Therefore in order to ensure correct protein folding it may be prudent to use at least partially a plant cystatin in plant systems. Thus, our site-directed modification of a plant proteinase inhibitor, and in particular a plant cystatin, and our hybrid proteinase inhibitor when including at least a part of a plant proteinase inhibitor have provided novel improved proteins for preferred, but not exclusive, use in plant systems.
It is of note that there is very little conservation in the alignment amino acid sequences shown in FIG. 1 above alignment amino acid 104 and therefore this makes our observation all the more startling. Previous site-directed modification studies of cystatins were concentrated on the highly conserved QVVAG motif referred to above, but modification of this region was always detrimental (12).
It follows from the foregoing that it is an object of the invention to provide a novel proteinase inhibitor having improved efficacy at least in terms of its binding to a proteinase.
It is also a further object of the invention to provide a novel proteinase inhibitor, ideally having improved efficacy, but also comprising a hybrid molecule which preferably, but not exclusively, comprises a part of a proteinase inhibitor from a first species and a part of proteinase inhibitor from a second species.
It is a further object of the invention to provide products including the whole or a part of the novel protein, or the whole or a part of the DNA encoding same, and also uses for this novel protein and/or DNA.
According to a first aspect of the invention there is therefore provided a proteinase inhibitor modified or manufactured so that it is more effective at inhibiting a proteinase with which it interacts than its corresponding naturally occurring counterpart.
Ideally, said proteinase inhibitor binds more strongly to the proteinase.
Accordingly there is provided a synthetic proteinase inhibitor which has a Ki with at least a 10 times lower value than its natural counterpart. For instance a change in Ki from 3xc3x9710xe2x88x928M to at least 3xc3x97109M represents such an improvement in Ki.
According to a second aspect of the invention there is provided a proteinase inhibitor including at least one site-directed amino acid deletion and/or substitution which lowers the Ki of the protein at least 10 fold.
Preferably the protein is a cystatin and ideally the site-directed deletion or substitution concerns deletion of either aspartic acid at position 86 of the amino acid sequence structure of Oc-I, or alternatively, deletion of aspartic acid at position 163 of the alignment amino acid sequence structure shown in FIG. 1 of an aligned proteinase inhibitor such as a cystatin, or alternatively, deletion of its functional counterpart in related proteinase inhibitors or cystatins.
Alternatively, the site-directed modification concerns substitution of said aspartic acid at said position for an alternative amino acid which has counter properties having regard to the functional property of the eliminated aspartic acid.
According to a third aspect of the invention there is provided the whole or part of the DNA sequence (SEQ ID NO:29) structure shown in FIG. 2 which DNA sequence structure encodes an example of a protein according to a first aspect of the invention.
All the proteins of the invention, or the corresponding DNA sequence structures have utility in combating diseases whose symptoms are at least partially caused by or characterised by proteinase production. Thus the novel proteins and corresponding DNA sequence structures can be used, directly or indirectly, to prevent, alleviate or mitigate such diseased conditions. For example, a host organism suffering from a pathogenic, pest or parasitic condition involving protein breakdown via proteinases can be treated by receiving at least one protein of the invention. Treatment can be undertaken by applying the said protein of the invention directly to the diseased organism, for example, in the form of a chemical agent such as a pesticide, fungicide etc. or a medicament, or alternatively, by introducing the genetic sequence structure for the said protein of the invention into the genome of the host organism and ensuring that the said inventive protein is expressed by the host organism which organism is then equipped to fight the disease.
In the foregoing paragraph any one or more of the proteins of the invention may be used as aforedescribed. For example, a selected combination of the proteins of the invention, that is to say proteins including site-directed modifications and/or hybrid proteins may be used to counter the effects of any one or more proteinases.
Ideally the transformed organism is a plant.
According to a further aspect of the invention there is provided a method of conferring resistance to proteolytic damage comprising modifying or transforming a host organism so that it expresses the protein of the invention.
According to a yet further aspect of the invention there is provided a construct including a whole or part of the DNA sequence structure of the invention. Said construct may include a plasmid or a vector.
According to a yet further aspect of the invention there is provided use of the protein or DNA sequence structure of the invention as a medicament to combat proteolytic conditions ideally of a pathogenic, parasitic or pest nature.
According to a further aspect of the invention there is provided a composition effective against pathogenic, parasitic or pest diseases including the protein of the invention.
According to a yet further aspect of the invention there is provided a transgenic plant transformed with DNA encoding the protein of the invention, and ideally the DNA shown in FIG. 2, which DNA is coupled to a suitable promoter sequence so that the protein of the invention can be expressed. Ideally, expression is either generally within the plant or in the locale of the pest, pathogen or parasite interaction with the plant. As examples reference 13 provides general methods for identifying promoters from the locale of a pest, pathogen or parasite of a plant. Alternatively, or in addition expression may be selected so as to occur at a selected given point in time.
Preferably said transformed plant is a cereal crop, vegetable crop, oil crop, sugar crop, forage or turf grass, fibre plant, herbalspice plant, fruit crop or indeed any decorative plant.
According to a yet further aspect of the invention there is provided a transformed organism, plant or otherwise, which includes DNA encoding the protein of the invention, and ideally the DNA shown in FIG. 2, so that said protein can be harvested for the purpose of providing sources thereof.
Preferably, said construct is provided with suitable promoters for ensuring expression of the protein of the invention.
According to a yet further aspect of the invention there is provided a method for controlling a pathogen, parasite or pest comprising exposing said pathogen, parasite or pest to the protein of the invention.
According to a further aspect of the invention there is provided use of the protein of the invention to control a pathogen, parasite or pest.
According to a vet further aspect of the invention there is provided any one or more of the primers shown in Table 2, or primers of similar nature having additions, deletions or modifications thereto which still enable the primers to function as described herein.
The modified proteinase inhibitors of the invention may also include novel combinations of proteinase inhibitors either derived from the same or different kingdom, phylum, class, order, family, genus or species. For example, fraction(s) of animal-derived proteinase inhibitor may be combined with fraction(s) of plant-derived proteinase inhibitor, all or one or more of which may or may not include the aforedescribed modification to improve efficacy. Or alternatively, different sorts or types of plant proteinase inhibitors may be combined to provide a novel plant proteinase inhibitor, or alternatively, different sorts or types of animal proteinase inhibitors may be combined to provide a novel proteinase inhibitor, all or one of more of which may or may not include the aforedescribed modification to improve efficacy.
According to a yet further still aspect of the invention there is provided a protein and/or sequence of DNA comprising a first part from a first proteinase inhibitor and at least one other part trom at least one other proteinase inhibitor.
In a preferred embodiment of the invention the DNA sequence of the further still aspect of the invention is provided in a construct so that a corresponding protein can be produced in target tissue such as host cell tissue.
According to a yet further aspect of the invention there is provided target tissue or host cell tissue transformed with the DNA sequence structure of the further still aspect of the invention.
According to a yet further aspect of the invention there is provided a protein comprising a first part from a first cystatin and at least one other part from at least one other cystatin.
In a preferred embodiment said first part of said DNA sequence or said protein comprises plant-derived cystatin DNA or protein respectively, and said at least one other part comprises animal-derived cystatin DNA or protein respectively.
Ideally said animal-derived DNA or protein corresponds to DNA or protein from the active site of animal-derived cystatin; and preferably said plant-derived cystatin DNA or protein corresponds to DNA or protein from a structural site or structural sites or said plant-derived cystatins.
Alternatively, said DNA sequence or protein comprises different sorts or types of plant-derived cystatins.
Alternatively again, said DNA sequence or protein comprises different sorts or types of animal-derived cystatin.
According to a yet further aspect of the invention there is provided protein and/or DNA sequence structure relating to a novel proteinase inhibitor comprising both the aforementioned hybrid proteinase inhibitor and also the aforementioned site-directed modification.
All of the proteinase inhibitors of the invention have application for countering the effects of proteinases and for use in methods relating to such effects.
Thus generally speaking the invention relates to the re-design of proteins which exhibit improved functional activities. Site-directed modifications or regions of amino acid sequence are replaced with either a corresponding region of a protein (from any organism) which exhibits the desired characteristics, or with designed synthetic sequences. The amino acid framework of the original protein is ideally maintained in the final hybrid molecule.